Separation of materials from a liquid dispersion by sedimentation

ABSTRACT

Sedimentation classification of particulate material in liquid dispersion. The dispersion is centrifuged in a tubular vessel comprising at least two neighboring compartments communicating via at least one opening which may be capillary. The lowermost compartment has a sealed bottom and the uppermost has an open upper end. Upon completion of the centrifugation a liquid fraction with dispersed particulate material is decanted from the uppermost compartment. Useful products may also be recovered from other compartments.

BACKGROUND OF THE INVENTION

The present invention concerns classification of particulate material inliquid dispersion by sedimentation so as to obtain at least twofractions of particulate material of different densities, and therecovery of at least one of such fractions. The sedimentation may beinduced by gravity by centrifugation and the liquid dispersion to beclassified in accordance with the present invention may be suspensionsor emulsions.

The present invention is applicable to a variety of purposes such asdiagnostic, analytical and preparative operations, typical applicationsbeing the separation of leucocytes from native blood, the splitting of arecovered leucocyte fraction into B-cells and T-cells, separation of anantigen/antibody complex produced in vitro in a reaction mixture fromother particulate material, classification of lipoproteins,classification of marrow cells and the like.

For certain centrifugation techniques there are commonly used testtube-like vessels so designed that they are capable of withstanding thehigh stresses prevailing during centrifugation. In such a vessel theinterface between layers resulting from centrifugation is a labilestructure and consequently special care has to be taken in order toavoid back-mixing of the fractions. This in turn gives rise to problemsin the separation of fractions from each other with the consequence thatsuch separation is a tedious and slow operation.

SUMMARY OF THE INVENTION

It is accordingly the object of the present invention to provide animproved method and means for the classification of particulate materialin liquid dispersion free of the shortcomings of the prior art.

In accordance with the present invention there is provided a method ofclassification by sedimentation of a particulate material in liquiddispersion, characterized by:

(i) providing a tubular vessel comprising at least two alignedcompartments with each two neighbouring compartments communicating viaat least one opening and having a first extreme compartment with asealed bottom and a second extreme compartment with an open upper end;

(ii) charging said liquid dispersion into said vessel;

(iii) subjecting the so charged vessel to centrifugation; and

(iv) decanting a liquid fraction with particulate material dispersedtherein from said second extreme compartment.

The opening may be in the form of a bore or hole or a plurality of boresand/or holes and the diameter thereof is not critical and will depend,inter alia, on the length of the hole/s or bore(s) and the nature of theparticulate material to be classified. It is preferred that the diametershould not exceed 10 mm and particularly preferred are openings having adiameter of not more than 5 mm., and the openings may be considerablynarrower, e.g. of a capillary nature.

During decantation of the liquid from the second extreme compartment,the entire vessel may be inverted without any danger of back-mixing.

The method according to the invention may be performed by gravitysedimentation or by centrifugation induced sedimentation.

If desired the method according to the invention may be performed withdensity adjustment prior to centrifugation, as known per se, e.g. by theaddition of a solute or of a working liquid of preselected density.Where a working fluid is used it may be of a uniform densityintermediary between the densities of particulate material fractions tobe classified, or have a density gradient also as known per se.

Where, in accordance with the invention only two particulate fractionsare to be separated from each other and only the lighter fraction is tobe recovered as, for example, in many instances of blood diagnosis whereonly the leucocytes, which are the lighter fraction, have to berecovered, the vessel may be made as one disposable unit comprising onlytwo compartments and be discarded after the recovery of the upperfraction as specified. Where, on the other hand, the dispersedparticulate material is to be fractionated into two or more fractions ofdifferent densities, each of which is to be recovered separately, thevessel is so designed that upon centrifugation the contents of eachcompartment may be recovered separately. When the vessel comprises onlytwo compartments the bottom portion of the first extreme compartment maybe removable and upon completion of centrifugation the contents of thesecond extreme compartment are recovered by decantation as specified,and the contents in the first extreme compartment are recovered byremoval of the bottom part and drainage.

Alternatively and where the dispersed particulate material is to beclassified into more than two fractions and the vessel comprisesaccordingly more than two compartments, the vessel may be in the form ofa disassemblable unit comprising two or more constituent parts each ofwhich corresponds to one compartment, and upon completion of thecentrifugation operation the vessel is disassembled and the contents ofeach compartment are recovered separately.

The invention further comprises a tubular sedimentation vessel forcarrying out the above method comprising at least two alignedcompartments with each two neighbouring compartments communicating viaat least one opening, having a first extreme compartment with a sealedbottom and a second extreme compartment with an open end.

It is preferred that the diameter of the openings should not exceed 10mm and particularly preferred are vessels as specified in which thediameter of the openings does not exceed 5 mm.

In accordance with one embodiment of a vessel according to theinvention, the vessel is in form of a disposable unit having twocompartments.

In accordance with another embodiment of a vessel according to theinvention, the bottom of the first extreme compartment is sealed bymeans of a removable bottom piece.

In accordance with yet another embodiment of a vessel according to theinvention, the vessel is disassemblable and comprises tubularconstituent parts, each corresponding to one compartment of theassembled vessel, each two neighbouring parts being adapted for tightinterengagement in that a downward protecting stud portion of one unitis adapted to be received in a tight fit by an open end of the otherunit.

The openings through which two neighbouring units in a vessel accordingto the invention communicate with each other may be provided by a boredpartition by which two neighbouring compartments are separated from eachother. Alternatively, it is also possible to use porous material.

In the following description the invention is described with referenceto centrifugation induced sedimentation, it being understood that it isnot limited thereto and applies also to gravity sedimentation.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated, by way of example only, in theaccompanying drawings in which:

FIGS. 1 (A)-(E) shows a prior art centrifugation separation scheme;

FIGS. 2 (A)-(D) shows a centrifugation separation scheme according tothe invention; and

FIGS. 3-6 show various embodiments of centrifugation vessels accordingto the invention.

DESCRIPTION OF THE PRIOR ART

The prior art centrifugation separation scheme shown in FIG. 1 concernsthe classification of blood for the separation of leucocytes anderythrocytes from each other and the recovery of the leucocytes.

As shown, a centrifugation tube 1 is charged with a polysaccharidesolution 2 which serves for density adjustment, and a blood portion 3 isthen charged on top of the polysaccharide solution 2. During chargingcare has to be taken that the blood 3 and the polysaccharide solution 2remain stratified without any undue mixing which might affect theclassification efficiency. Upon centrifugation the material insidevessel 1 becomes stratified with the formation of a lower clot 4 oferythrocytes, an intermediary body of liquid 5 consisting ofpolysaccharide solution and an upper body of liquid 6 consisting mainlyof plasma with an agglomerate of leucocytes 7 floating near theinterface with body 5. For the recovery of the leucocytes 7 the upperliquid body 6 has to be sucked out very carefully by means of a pipette8 and transferred into a different vessel 9. The suction operation hasto be done very carefully in order to avoid back-mixing with theerythrocytes in clot 4. Because of this, the separation of the upperliquid body 6, 7 is a tedious operation and cannot be carried out tocompletion.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The centrifugation separation according to the invention showndiagrammatically in FIG. 2, also concerns the classification of bloodinto leucocytes and erythrocytes with the aid of density adjustment bymeans of a polysaccharide solution. As shown, tubular vessel 10comprising a lower compartment 11 and an upper compartment 12 separatedfrom each other by a partition in form of a double concave block 13 witha central capillary bore 14, is charged with a polysaccharide solution15 such that the solution reaches the upper end of the capillary bore14. Thereafter the vessel 10 is charged with a blood portion 16 which ispoured into the upper compartment 12. No special care has to be takenduring this charging operation because block 13 with the capillary bore14 prevent any significant mixing of the body of solution 15 and theblood portion 16. Upon centrifugation the material inside vessel 10stratifies into a lowermost erythrocyte clot 17 with a small body 18 ofpolysaccharide solution on top, an intermediary body 19 ofpolysaccharide solution, an upper body 20 of plasma with an agglomerateof leucocytes 21 floating therein near the interface with body 19. Forthe recovery of the leucocytes 21 the entire contents of the uppercompartment 12 is poured into another vessel 22 by decantation as shownon the righthand side of FIG. 2. During this decantation the capillarybore 14 ensures that none of the clot of erythrocytes 17 escapes fromcompartment 11 whereby any back-mixing of the erythrocytes andleucocytes is prevented.

It is assumed that the erythrocytes are not required in this particularoperation and consequently the vessel 10 is designed as a disposableunit which is discarded after the contents of the upper compartment 12has been poured into vessel 22 as specified.

The sedimentation vessel according to the invention used in thecentrifugation separation scheme according to FIG. 2 is shown once morein FIG. 3 where the same numerals are used as in FIG. 2. As shown,vessel 10 comprises a lower compartment 11 and an upper compartment 12separated from each other by a double concave block 13 having a centralcapillary bore 14 through which compartments 11 and 12 communicate witheach other. The diameter of bore 14 may not exceed 5 mm while the lengththereof is not critical. The double concave design of block 13 ensuresthat no air pockets are formed in the lower compartment 11 and that asump is formed in the upper compartment 12 for the complete drainageduring centrifugation of the denser fraction to bore 14. The vessel ofFIG. 3 is designed to be disposable.

The embodiment of the vessel according to the invention shown in FIG. 4is designed for repeated use and it enables the separate recovery ofboth the upper and lower fractions. As shown, the vessel 23 isessentially of a design similar to that of FIG. 3. It comprises a lowercompartment 24 and an upper compartment 25 separated from each other bya partition in form of a double concave block 26 having a centralcapillary bore 27.

The lower compartment 24 is formed with a removable bottom piece 28having a circumferential recess 29 adapted to receive in a tight fit adownwardly projecting stud portion 30 of the main body of vessel 23.

The embodiment of FIG. 4 is used where two sedimentation fractions areeach to be recovered. Accordingly, after centrifugation the contents ofthe upper compartment 25 are withdrawn by decantation similarly as shownin FIG. 2 and thereafter the bottom piece 28 is removed whereupon thecontents of the lower compartment 24 are drained off.

The embodiment of FIG. 5 is again designed for repeated use and itcomprises two constituent parts, each corresponding to one compartmentof the vessel. As shown the vessel 31 comprises a lower unit 32 and anupper unit 33. The lower unit 32 has an upper, open end portion 34 whilethe upper unit 33 has a downwardly projecting stud portion 35 fittedwith a capillary bore 36 and adapted to be received in a tight fit bythe upper end portion 34 of the lower unit 32. Stud portion 35 alsoforms the block that separates units 32 and 33 from each other andsimilar as in the embodiment of FIG. 4 it is of a double concave shape.

The embodiment of FIG. 5 is also used where two fractions are each to berecovered. Accordingly after centrifugation the contents of the upperunit 33 are removed by decantation in a similar way as shown in FIG. 2.When this decantation is complete the upper unit 33 is removed wherebythe fraction that has collected in the lower chamber 32 becomesaccessible.

The embodiment of the centifugation vessel 37 shown in FIG. 6 comprisesthree constituent units 38, 39 and 40. The lowermost unit 38 is of asimilar design as unit 32 in FIG. 5 and it comprises an upper, open endportion 41. The intermediary unit 39 comprises a downward projectingstud portion 42 having a central capillary bore 43 of double concaveshape similar as stud 35 of FIG. 5. The intermediary unit 39 furthercomprises an upper open end portion 44 and the upper unit 40 comprises adownward projecting double concave stud portion 45 fitted with acentral, capillary bore 46.

In the assembled state the stud portion 42 of the intermediary unit 39is received in a tight fit by the open end portion 41 of the lower unit38 and likewise the downward projecting stud portion 45 of the uppermostunit 40 is received in a tight fit by the open end portion 44 of theintermediary unit 39.

The embodiment of FIG. 6 is used for the separate recovery of threefractions: After separation of the uppermost fraction in unit 40 bydecantation, unit 40 is removed and the intermediary fraction that hascollected in unit 39 is separated again by decantation, whereupon thatunit is also removed leaving the third, lowermost fraction accessible inunit 38.

WORKING EXAMPLES EXAMPLE 1

Separation of Mononuclear Cells from Blood

1.3 mm of an aqueous polysaccharide solution (d=1.077) available underthe trade mark Histopaque from Sigma Chemical Co. of St. Louis, Mo.,U.S.A. are poured into the lower compartment 11 of a vessel according toFIG. 3 so that the upper meniscus of the liquid is at the top of thebore 14. 2.0 ml blood are poured into the upper chamber of the vesselwithout any special precaution being required to avoid mixing of theblood with the polysaccharide solution, such mixing being essentiallyprevented by the special design of the vessel. The vessel with itscontents is then centrifuged at 750 xg for a period of 20 minutes. Aftercentrifugation the upper compartment 12 contains three layers, an upperlayer of plasma, a white interface layer containing the mono-nuclearcells and a lower layer containing the said polysaccharide solution. Thelower compartment 11 contains a layer of the polysaccharide solution andat the bottom a clot of red blood cells. The entire contents of theupper compartment 12 are poured into another vessel with no specialprecautions being required, any such backmixing being preventedautomatically by block 13 and capillary bore 14. Phosphate buffersolution (PBS) is added to the mononuclear cell suspension recoveredfrom the upper compartment 12 and that suspension is then processed asmay be required.

EXAMPLE 2

Demonstration of Improved Separation Efficiency

The improvement of the efficiency of separation in accordance with theinvention was demonstrated as follows: 59 samples of blood wereprocessed in parallel using respectively the prior art method asillustrated in FIG. 1 and the method according to the invention asillustrated in FIG. 2 and described in Example 1. Protein content of theresultant mononuclear cell suspension was assayed according to themethod of Lowry. The results of this comparative study are summarized inthe table below:

    ______________________________________                                        Method  Number of Samples                                                                            Yield*  Efficiency factor                              ______________________________________                                        Prior art                                                                             59             0.297   1.00**                                         method                                                                        Method  59             0.367   1.24                                           according                                                                     to the                                                                        invention                                                                     ______________________________________                                         *mg protein in mononuclear cell suspension derived from 1.0 ml blood          (mean).                                                                       **By definition.                                                         

The advantages which are obtainable in accordance with the invention ascompared to the prior art may be summed up briefly as follows:

(i) Charging in case of density adjustment by means of solutions:whereas in accordance with the prior art charging must be effected verycarefully in order to avoid initial mixing of the sample with thedensity adjustment solution, in accordance with the invention suchmixing is essentially prevented and accordingly no precaution has to betaken during sampling.

(ii) Removal of an upper fraction: whereas in accordance with the priorart upon sedimentation an upper fraction can be removed only by suctionand special care has to be taken to avoid any back-mixing, in accordancewith the invention an upper fraction is poured out of the uppercompartment by decantation and any back-mixing is inherently prevented;

(iii) Handling of vessel after sedimentation: whereas in accordance withthe prior art handling of the sedimentation tube after sedimentation isrestricted and should be done only with the greatest care, there are nolimitations on the handling of the sedimentation vessel in accordancewith the present invention.

(iv) Simultaneous operation: whereas in accordance with the prior artone operator can only handle one sedimentation tube at a time with theconsequence that recovery of upper fractions from a plurality ofsedimentation tubes can only be effected sequentially, in accordancewith the invention a plurality of sedimentation vessels can be handledsimultaneously, e.g. by placing them in a rack and inverting the rack soas to pour out simultaneously the contents of all the uppercompartments.

(v) Efficiency: whereas in accordance with the prior art the recoveryyields of upper fractions are inherently incomplete because of thedanger of backmixing during suction, in accordance with the presentinvention an upper fraction can be recovered in toto without any dangerof back-mixing whereby the separation efficiency is increased by 20-25%.

I claim:
 1. A method of classification by centrifugation of particulatematerial in liquid dispersion with at least two fractions, characterizedby:(i) providing a tubular vessel comprising at least two alignedcoaxial compartments with each two neighbouring compartmentscommunicating via at least one opening, said tubular vessel having afirst extreme compartment with a sealed bottom and a second extremecompartment with an open end; (ii) charging a working liquid into saidtubular vessel via said second extreme compartment thereof so as to fillcompletely said first extreme compartment, said working liquid having adensity intermediate the densities of said at least two particulatematerial fractions to be classified; (iii) charging said particulatematerial in liquid dispersion into said vessel via said second extremecompartment; (iv) subjecting the so-charged vessel to centrifugation,thereby to cause a heaviest fraction of said particulate material tomigrate axially into said first extreme compartment; and (v) decanting aliquid fraction with particulate material dispersed therein from saidsecond extreme compartment.
 2. A method according to claim 1 whereinsaid working liquid has a density gradient.
 3. A method according toclaim 1 comprising using a vessel with only two compartments, aftersedimentation recovering only the contents of the second extremecompartment and then discarding said vessel.
 4. A method according toclaim 1 comprising using a vessel with only two compartments, sodesigned that said first extreme compartment is adapted for the recoveryof its contents, and after sedimentation recovering the contents of eachcompartment.
 5. A method according to claim 1 comprising using adisassemblable vessel with two or more compartments, disassembling thevessel after sedimentation and recovering the contents of eachcompartment.
 6. The method of claim 1, wherein said working liquid ismiscible with a liquid phase of said dispersion.
 7. A tubularcentrifugation vessel for carrying out a method of classifying bycentrifugation a particulate material in liquid dispersion, comprisingatleast two axially-aligned compartments with each two neighbouringcompartments communicating via at least one opening, said tubularcentrifugation vessel having a first extreme compartment with a sealedbottom and a second extreme compartment with an open end, said at leastone opening being of a size to admit in the course of centrifugation thepassage of a fraction of the particulate material in liquid dispersionthat is being centrifuged, and not admitting passage of the particulatematerial in liquid dispersion under normal gravitational conditions whena working liquid is charged to a level of said at least one opening,thereby forming a meniscus.
 8. A vessel according to claim 7 having twocompartments.
 9. A vessel according to claim 8 having a removablebottom.
 10. A vessel according to claim 7 being disassemblable andcomprising tubular constituent parts, each corresponding to onecompartment of the assembled vessel, each two neighbouring parts beingadapted for tight interengagement in that a downwardly projecting studportion of one unit is adapted to be received in a tight fit by an openend of the other unit.
 11. A vessel according to claim 7 comprising apartition between every two neighbouring compartments, having at leastone opening.
 12. A vessel according to claim 7 wherein the diameter ofany opening does not exceed 10 mm.
 13. A vessel according to claim 12wherein the diameter of any opening does not exceed 5 mm.
 14. A vesselaccording to claim 7 wherein the opening is of a capillary nature.
 15. Avessel according to claim 7 wherein every two compartments are separatedby porous material.
 16. The tubular vessel of claim 7, additionallycomprisinga partition situated in said vessel and separating eachcompartment from the other compartment, and said at least one openingbeing a capillary bore extending through said partition.
 17. Thecombination of claim 16, wherein said partition is in the form of adouble concave block.
 18. The combination of claim 16, additionallycomprisinga removable bottom piece enclosing at least a part of saidfirst compartment, whereby contents of the same can be drained off. 19.The combination of claim 18, wherein said bottom piece comprises acircumferential recess and a remainder of said vessel comprises aprojecting stud,with said stud and recess formed to engage in a tightfit.
 20. The combination of claim 18, whereinsaid removable bottom piececomprises an open upper end, and said partition comprises a projectingstud portion, with said stud portion and upper end of said bottom pieceformed to engage in a tight fit.
 21. The combination of claim 20,additionally comprisinga third compartment aligned with said first twocompartments, a second partition situated to separate said second andthird components, and a second capillary bore extending through saidsecond partition and through which said second and third compartmentscommunicate, wherein said second partition comprises a projecting studportion formed to engage said open end of said second compartment in atight fit, and said second and third compartments are separable from oneanother.